Functionalized active-nucleus complex sensor

ABSTRACT

A functionalized active-nucleus complex sensor that selectively associates with one or more target species. The functionalized active-nucleus complex comprises an active-nucleus and a targeting carrier. The targeting carrier comprises a first binding region having at least a minimal transient binding of the active-nucleus to form the functionalized active-nucleus complex that produces a detectable signal when the functionalized active-nucleus complex associates with the target species and a second binding region that selectively associates with the target species. Included is a method for assaying and screening for one or a plurality of target species utilizing one or a plurality of functionalized active-nucleus complexes with at least two of the functionalized active-nucleus complexes having an attraction affinity to different corresponding target species. The method comprises the steps of functionalizing an active-nucleus, for each functionalized active-nucleus complex, by incorporating the active-nucleus into a macromolucular or molecular complex that is capable of binding one of the target species. Then bringing the macromolecular or molecular complexes into contact with the target species and detecting the occurrence of or change in a nuclear magnetic resonance signal from each of the active-nuclei in each of the functionalized active-nucleus complexes in order to either monitor the occurrence of binding between each of the functionalized active-nucleus complexes and the target species or monitor a subsequent change in the environment of the target species after the binding occurs.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/903,279 filed on Jul. 11, 2001, now U.S. Pat. No. ______, whichclaims priority to U.S. provisional application, serial No. 60/218,549filed on Jul. 13, 2000. Priority is claimed to both applications, andboth applications are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under ContractNo. DE-AC03-76SF00098 awarded by the Director, Office of EnergyResearch, Office of Basic Energy Sciences, Materials Sciences Division,Physical Biosciences Division, of the U.S. Department of Energy.Additionally, support was provided by the National Science FoundationPre-Doctoral Fellowship Program. The Government has certain rights inthis invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

[0003] Not Applicable

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] A molecular or macromolecular structure and method of use aredisclosed in which an active-nucleus is functionalized in at least atransient interaction with a target carrier to form a sensor thatselectively associates with a target substrate or environment to producea detectable signal. More specifically, a functionalized active-nucleuscomplex sensor is described in which an active-nucleus gas such ashyperpolarized xenon, hyperpolarized helium, or sulfur hexafluoride, oractive-nuclei ¹⁹F derivatives are bound in a carrier structure having abinding region specific for a target species. Upon binding to the targetspecies the active-nucleus produces a detectable nuclear magneticresonance signal or is detectable as a magnetic resonance imagingcontrast agent. A plurality of target specific sensors may be utilizedin the assaying and screening of samples containing the plurality oftargets under either in vivo or in vitro conditions.

[0006] 2. Description of the Background Art

[0007] The detection of biological molecules and their interactions is asignificant component of modern biomedical research. In currentbiosensor technologies, simultaneous detection is limited to a smallnumber of analytes by the spectral overlap of their signals. Recentbiosensor technologies exploit surface plasmon resonance (1),fluorescence polarization (2), and fluorescence resonance energytransfer as detection methods (3). Although the sensitivity of suchtechniques is excellent, it has proven challenging to extend theseassays to multiplexing capabilities because of the difficulty indistinguishing signals from different binding events. While nuclearmagnetic resonance (NMR) spectroscopy is able to finely resolve signalsfrom different molecules and environments, the spectral complexity andlow sensitivity of NMR spectroscopy normally preclude its use as adetector of molecular targets in complex mixtures. Notable successes(4,5) in the application of NMR to such problems are still limited bylong acquisition times or a limited number of detectable analytes. Laserpolarized xenon NMR benefits from good signal to noise and spectralsimplicity with the added advantage of substantial chemical shiftsensitivity.

[0008] U.S. Pat. No. 5,642,625 discloses a high volume hyperpolarizerfor spin-polarized noble gas. A method and apparatus are presented thatallow spin exchange between atoms of the noble gas and an alkali metalsuch as rubidium.

[0009] Described in U.S. Pat. No. 5,785,953 is a magnetic resonanceimaging technique using hyperpolarized noble gases as contrast agents.In particular, hyperpolarized xenon and helium are utilized in spatialdistribution studies.

[0010] The foregoing references/patents reflect the state of the art ofwhich the applicant is aware and are tendered with the view towarddischarging applicant's acknowledged duty of candor in disclosinginformation which may be pertinent in the examination of thisapplication. It is respectfully submitted, however, that none of thesereferences/patents teach or render obvious, singly or when considered incombination, applicant's claimed invention.

SUMMARY OF THE INVENTION

[0011] An object of the present invention is to disclose a sensor andmethod of use comprising an active-nucleus (guest) and target carrier(host) that generates an NMR and/or MRI detectable signal uponassociation with a biological target.

[0012] Another object of the present invention is to relate a biosensorand method of in vivo and in vitro assaying/screening use that comprisesa functionalize active-nucleus complex that selectively binds to andsignals the presence of a desired biological target species.

[0013] A further object of the present invention is to describebiosensors and methods of in vivo and in vitro assaying/screening usethat comprises a plurality of functionalize active-nucleus complexeswith each complex selectively binding to and signaling the presence of adesired biological target species or analyte.

[0014] Still another object of the present invention is to present abiosensor and method of use in which the biosensor comprises anactive-nucleus bound to a target carrier in which when the targetcarrier binds to a target species/analyte a detectable signal isproduced upon the binding or upon alterations in the targetspecies/analyte or its environment after the binding.

[0015] Yet a further object of the present invention is to disclose aplurality of biosensors and a multiplexed method of use in which each ofthe biosensors comprises an active-nucleus bound to a target carrier inwhich when the target carrier binds to a target species/analyte adetectable signal is produced upon the binding or upon alterations inthe target species/analyte or its environment after the binding, whereinall the biosensors' signals are simultaneously detectable.

[0016] Disclosed is a novel, functionalized active-nucleus sensor orbiosensor that is directed to and signals the presence of a desiredbiological target species, often of biological origin or importance. Anactive-nucleus that presents a detectable signal to either nuclearmagnetic resonance (NMR) or magnetic resonance imaging (MRI) techniquesis utilized in conjunction with a target specific carrier that interactswith both the active-nucleus and a biological target substrate orenvironment. The active-nucleus is capable of at least a minimaltransient binding to a targeting carrier. The targeting carrierassociates with the target substrate or environment, thereby stimulatingthe production of or change in the detectable signal from theactive-nucleus in a functionalized interaction. “Functionalized” impliesthat when the active-nucleus is bound, in at least a minimal transientmanner, by the targeting carrier, that the active-nucleus then respondsto and signals the association between the targeting carrier and thetarget substrate or environment.

[0017] Since the basic subject invention enables the creation of severalextremely powerful and versatile sensors and techniques that have eludedresearchers for many years, a number of related embodiments aredisclosed below. One requirement for the subject invention is that thereporter nucleus be sufficiently “active” or capable of producing asignal that is detectable by NMR or MRI techniques. Hyperpolarized noblegases such as xenon and helium meet this requirement, as do other nucleisuch as ¹⁹F, if present in sufficiently high concentrations. Thus,“active” implies that the nucleus generates a suitable signal that iscapable of detection by NMR (either in strong or weak magnetic fields)and/or MRI contrast procedures. Several relatively standard techniquesnow exist for hyperpolarizing noble gases and include optical pumping orspin exchange procedures.

[0018] It is important to appreciate that for the subject invention thesignal produced by the functionalized active-nucleus is studied directlyto follow the behavior of the biological target substrate orenvironment. For example, xenon (as indicated above, other suitableactive-nuclei are also contemplated as being within the realm of thisdisclosure), has a chemical shift that is enormously sensitive to itslocal chemical environment. With the large xenon NMR signal created byoptical pumping, the chemical shift can easily serve as a signature forthe different chemical surroundings in which the xenon is found. Directinteraction between xenon and a target molecule has been observed bymeasuring the chemical shift and relaxation properties on xenon (inparticular see, S. M. Rubin, M. M. Spence, B. M. Goodson, D. E. Wemmer,A. Pines, Proceedings of the National Academy of Sciences of the UnitedStates of America 97, 9472-9475 (2000) that was part of the ProvisionalApplication to which this application claims priority). However, theobservation of this direct contact may be limited by the weak binding ofxenon (or other suitable active-nuclei) to many target molecules ofinterest. To enhance the binding of the xenon, for example, to thebiological target species/substrate/molecule/analyte of interest, andthus the population of xenon in contact with the targetspecies/substrate/molecule/analyte, the xenon can be functionalized tostrongly bind to the biological targetspecies/substrate/molecule/analyte. This can be achieved by placing thexenon, or other suitable active-nuclei, in a target carrier thatchemically recognizes and binds to the target. The target carrier has afirst binding region that binds the xenon for at least a minimaltransient period (“minimal” in the sense of a sufficiently long periodof time to produce a useful signal) or, preferably, very strongly bindsthe xenon, and can not itself quickly relax the xenon polarization.Amplification of the sensing for both xenon and helium may be achievedby utilizing a “pool” of hyperpolarized active-nuclei atoms that eithersense the environment by changes in the functionalized active-nucleuscarrier complex (molecule, supramolecular, or microbubble environment)or else are in sufficiently rapid chemical exchange with active-nucleiin biosensor sites that are so sensitive, thereby amplifying thedetection intensity yet further.

[0019] The target carrier has a second binding region that binds to orreacts with the target species/substrate/molecule/analyte. The targetcarrier allows xenon, or other active-nuclei, to be held in closeproximity to the desired target, giving rise to a signal at adistinctive frequency indicating the presence of the targetspecies/substrate/molecule/analyte. The functionalizedactive-nucleus/target carrier complex can “recognize” any one of a widevariety of biological target species/substrates/molecules/analytes(virtually an unlimited set of organic/biomolecular structures)including biologically important species such as proteins, nucleicacids, carbohydrates, lipids, metabolites, and the like in either an invitro or non-invasive in vivo setting at either high or low NMR utilizedfield strengths. For example, with diseases, the diagnostic power of thesubject invention is quite clear. Various diseases presentcharacteristic/defining targets such as unusual membrane proteins,lipids, or carbohydrates, unusual analytes in body fluids, and the likewhose presence can easily be detected with the subject invention.

[0020] The subject method of assaying and screening for target speciesin in vivo and in vitro samples/subjects has many strengths, includingthe large signal to noise ratio afforded by the high polarizationachieve with hyperpolarization of xenon, helium, and other suitablenuclei. With xenon, for example, there is a negligible natural presenceof xenon, so there would be no interference from background xenonsignals. In contrast to fluorescence (and other techniques that generateoverlapping or interfering detection signals) assays and screeningprocedures, multiple functionalized active-nuclei tests are possible inone system (test-tube, plate, microplate, and the like), by creatingactive-nuclei carriers targeting differentspecies/substrates/molecules/analytes or by altering the structure ofthe probe itself or both (see below). Each target would give rise to aseparate active-nucleus chemical shift. These assays and screeningscould also be carried out non-invasively in vivo, avoiding the exposureto radiation that radiometric assays and screenings require. In the caseof optical pumping to create hyperpolarization of xenon and helium,because the large active-nuclei signals are generated by the opticalpumping, high magnetic fields are unnecessary (as mentioned previously),and the chemical shifts can be detected in low magnetic fields using asuperconducting quantum interference device (SQUID).

[0021] A preliminary calculation was performed (verified by the resultsobtained in Experimental Example #1, found below) to explore the initialfeasibility of the subject technique in vivo. Based on capabilities ofcurrent spectroscopy, 200 nanomoles of nuclear spins are necessary tomeasure a signal. To compete with or match other forms of assays orscreening procedures, 20 picomoles of target species must be detectable,A factor of 10⁴ in signal is required. The hyperpolarization compensatesfor at least a factor of 10³, and the additional factor of ten is gainedby the relatively simplicity of the spectrum, contrasted with a target(protein and the like) spectrum.

[0022] In its most basic configuration the subject invention comprisesan active-nucleus and a target carrier that associates with both theactive-nucleus and a desired target species to produce an detectablecharacteristic signal (typically a chemical shift or relaxation time forNMR or a contrast capability for MRI). The functionalized active-nucleuscomplex or subject biosensor that may comprise one or more identical orvaried second binding regions. Additionally, the functionalizedactive-nucleus complex or subject biosensor may have varied firstbinding regions. Also, both the first and second binding regions couldbe varied within the same subject biosensor. As indicated, the subjectinvention allows a huge array of possible targetspecies/substrates/molecules/analytes to be assayed/screened for in aparallel or multiplexing detection style within a single sample/subject.

[0023] Several possible active-nuclei gases exist, preferablehyperpolarized xenon and hyperpolarized helium, however, ¹⁹F and similarnuclei, in sufficient concentration, are also contemplated. Withfluorine atoms, an exemplary functionalized sensor comprises a targetcarrier having multiple fluorines such as a polyfluorinated dendrimerthat selectively binds an organic targetspecies/substrate/molecule/analyte or a form of fluorine such as sulfurhexafluoride trapped/bound within a functionalized (target specificbinding) enclosing structure such as in “bubble” or “microbubble”environment as exemplified by a liposome, micelle, vesicle, bucky-balltype structures, natural and synthetic polymeric cages, and like.Conformational changes or alterations in the effective pressure on the“bubble” or “microbubble” would induce detectable signal variations fromthe subject biosensor. Variations in the immediate vicinity orenvironment of the biosensor should be detectable and include changes inion concentrations, functioning of an ion channel, oxygenlevels/distribution, neuron activity, and the like. It is noted thathyperpolarized xenon and hyperpolarized helium will also function as thesignal reporting active-nuclei within similar functionalized “bubble” or“microbubble” structures.

[0024] The first binding region of the targeting carrierinteracts/associates/binds with the active-nucleus. This first bindingregion includes structures such as monoclonal antibodies, dendrimers,self-assembled lipid complexes, liposomes, cyclodextrins, cryptands,carcerands, microbubbles, micelles, vesicles, molecular tennis balls,fullerenes, many general cage-like structures, and the like.

[0025] The second binding region in the targeting carrier comprises thatportion of the subject biosensor that interacts with the targetspecies/substrate/molecule/analyte. It is noted that multiple secondbinding regions are contemplated and may be identical or varied forattachment to a plurality of target sites.

[0026] The basic subject biosensor may contain additional usefulcomponents/structures. One or more “tether” regions may be included andserve to separate the first and second binding regions and to permit aregion that may be further derivatized with additional moieties such assolubilizing regions. The solubilizing regions may contain polypeptides,carbohydrates, and other species that aid in solubilizing the subjectprobe.

[0027] More specifically, a functionalized active-nucleus biosensor isdisclosed that capitalizes on the enhanced signal to noise, spectralsimplicity, and chemical shift sensitivity of suitable active-nucleigases (for example only and not by way of limitation, hyperpolarizedxenon, hyperpolarized helium, and sulfur hexafluoride) andpolyfluorinated containing species (utilized to target organicmolecules) to detect specific targets. One subject sensor embodimentutilizes laser polarized xenon “functionalized” by a biotin-modifiedsupramolecular cage, including a tether region having a solubilizingregion, to detect biotin-avidin binding. This biosensor methodology canbe used in analyte assays and screening procedures or extended tomultiplexing assays for multiple analytes of screenings for multiplespecies.

[0028] Other objects, advantages, and novel features of the presentinvention will become apparent from the detailed description thatfollows, when considered in conjunction with the associated drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a schematic model showing a first embodiment of thesubject sensor illustrating a first binding region for holding theactive-nucleus and a second binding region that associates with thetarget.

[0030]FIG. 2 is the schematic model sensor shown in FIG. 1 with a targetspecies bound to the second binding region.

[0031]FIG. 3 is a schematic model showing a second embodiment of thesubject sensor illustrating a first binding region identical to thatdepicted in FIG. 1 and a varied second binding region.

[0032]FIG. 4 is a schematic model showing a third embodiment of thesubject sensor illustrating a varied first binding region and a secondbinding region identical to that depicted in FIG. 1.

[0033]FIG. 5 is a schematic model showing a fourth embodiment of thesubject sensor illustrating both a varied first binding region and avaried second binding region, relative to those seen in FIG. 1.

[0034]FIG. 6 is a schematic model showing a fifth embodiment of thesubject sensor illustrating a first binding region and a plurality ofvaried second binding regions.

[0035]FIG. 7 is a schematic model showing a sixth embodiment of thesubject sensor illustrating a polyfluorinated first binding region and asecond binding region that associates with an organic target species.

[0036]FIG. 8 is a schematic model showing a seventh embodiment of thesubject sensor illustrating a first binding region containing sulfurhexafluoride and a second binding region.

[0037]FIG. 9A is a schematic model showing an eighth embodiment of thesubject sensor illustrating a first binding region for holding theactive-nucleus, a second binding region that associates with the target,and a tether region that connects the first and second binding regions.

[0038]FIG. 9B is the eighth sensor embodiment, seen in FIG. 9A, bound toa target species.

[0039]FIG. 10 is the eighth sensor embodiment, seen in FIG. 9A,illustrating an active-nuclei exchange process that enhances thegenerated detection signal.

[0040]FIG. 11 is a specific chemical structure of the subject sensor,without an active-nucleus, showing a first binding region(cryptophane-A) for holding the active-nucleus, a second binding regionthat associates with the target, a tether region that connects the firstand second binding regions, and a solubilizing polypeptide attached tothe tether.

[0041]FIG. 12 is the specific chemical structure shown in FIG. 11 withthe active-nucleus xenon included within the cage-like, cryptophane-A,first binding region.

[0042]FIG. 13 shows ¹²⁹Xenon NMR spectra that monitors the binding of abiotin-functionalized xenon biosensor to avidin.

[0043]FIG. 14 shows the effect of cage structure (cryptophane-A in thetop “A” view and cryptophane-E in the bottom “B” view) on the boundxenon chemical shift.

[0044]FIG. 15 is a schematic diagram showing multiplexing withfunctionalized xenon biosensors in which the top spectrum shows thethree distinct functionalized xenon peaks, corresponding to differentfirst binding region cages tethered to three second binding regionligands. The bottom spectrum shows the effect of adding thefunctionalized xenon biosensor to an unknown sample solution havingtargets.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0045] There are several preferred embodiments of the subject inventiondisclosed in the specification and depicted in FIGS. 1-15. In thesubject invention's most basic configuration the subject inventioncomprises an active-nucleus (NMR or MRI detectable nuclei, preferablyhyperpolarized xenon or hyperpolarized helium, however, ¹⁹F is useful ifpresent at sufficient levels) and a target carrier that associates withboth the active-nucleus and a desired target to produce an detectablecharacteristic signal (typically a chemical shift or relaxation time forNMR or a contrast capability for MRI). FIG. 1 depicts the basicbiosensor (the functionalized active-nucleus complex) 5 configurationand FIG. 2 shows the basic functionalized biosensor 5 bound to a targetspecies/substrate/molecule/analyte 25. An active-nucleus 10 is bound ina targeting carrier. The targeting carrier comprises, at least, a firstbinding region 15, binding the active-nucleus, and a second bindingregion 20, wherein the second binding region 20 has a binding affinityfor the target species/substrate/molecule/analyte 25 (the dashed lineindicating the binding domain in the target). The detectable signalgenerated from the bound complex 5 in FIG. 2 is distinguishable from thedetectable signal produced from the unbound complex 5 in FIG. 1 (seeFIG. 13 for an equivalent signal shift).

[0046] As indicated above, one extremely useful characteristic of thesubject invention is that the signal produced from the subject sensor ishighly dependent upon its immediate environment and that signals createdfrom similar, but not identical, sensors can be distinguished andutilized to detect multiple target species/substrates/molecules/analyteswithin the same sample. For example, FIG. 3 depicts a functionalizedactive-nucleus complex or subject biosensor 5′ that has a varied secondbinding region 21, relative to the second binding region 20 seen inFIGS. 1 and 2. Thus, biosensor 5′ would bind to a different targetspecies/substrate/molecule/analyte or a different location on theoriginal target species/substrate/molecule/analyte 25.

[0047] Additionally, FIG. 4 illustrates a functionalized active-nucleuscomplex or subject biosensor 5″ that has a varied first binding region16, relative to the first binding region 15 seen in FIGS. 1 and 2. Thus,biosensor 5″ would generate a different signal than the signal producedby biosensor 5.

[0048] Also, both the first 16 and second binding regions 21 could bevaried, relative to the biosensor seen in FIGS. 1 and 2, within the samesubject biosensor to form biosensor 5′″, as seen in FIG. 5. Asindicated, the subject invention allows a huge array of possible targetspecies/substrates/molecules/analytes to be assayed/screened for in aparallel or multiplexing detection style within a single sample.

[0049]FIG. 6 illustrates a subject biosensor that has several differentsecond binding regions 20, 21, 22, and 23 attached to a first bindingregion producing sensor 5″″. Sensor 5″″ may bind to one or more targetsvia the presented second binding regions.

[0050] As indicated, several possible active-nuclei gases exist for anytarget species, preferable hyperpolarized xenon, hyperpolarized helium,and sulfur hexafluoride, however, ¹⁹F, in sufficient concentration, isalso contemplated for organic/biological targets. With fluorine atoms,an exemplary functionalized sensor comprises a target carrier havingmultiple fluorines such as a polyfluorinated dendrimer that selectivelybinds an organic/biological target species/substrate/molecule/analyte,as seen in FIG. 7. The polyfluorinated first binding region 35 may be adendrimer or other suitable structure, including, but not limited tonatural and synthetic polymers and the like. Additionally, sufficientfluorine to produce an acceptable signal may be in the form of fluorinein sulfur hexafluoride and similar compounds. FIG. 8 illustrates sulfurhexafluoride 45 trapped/bound within a functionalized (target specificbinding) enclosing structure 40 such as in “bubble” or “microbubble”environment as exemplified by a liposome, micelle, vesicle, bucky-balltype structures, natural and synthetic polymeric cages, and the like. Asecond binding region 20 is coupled to the enclosing structure 40 andbinds the target. Conformational changes or alterations in the effectivepressure on the “bubble” or “microbubble” would induce detectable signalvariations from the active-nucleus in a subject biosensor. Variations inthe immediate vicinity of the biosensor should be detectable and includesuch changes as: ion concentrations, oxygen levels, neuron activity, andthe like. It is noted that hyperpolarized xenon and hyperpolarizedhelium will also function as signal reporting active-nuclei withinsimilar functionalized “bubble” or “microbubble” structures.

[0051] It is noted that the subject targeting carrier comprises thefirst binding region (15 and 16 in FIGS. 1-6) thatinteracts/associates/binds with the active-nucleus. This first bindingregion includes structures such as monoclonal antibodies, dendrimers,self-assembled lipid complexes, liposomes, cyclodextrins, cryptands,cryptophanes, carcerands, microbubbles, micelles, vesicles, moleculartennis balls, fullerenes, many general cage-like structures, and thelike. As long as structure or chemical nature of the first bindingregion permits effective signal producing interactions with theactive-nucleus and binding to the target is not negated, a wide range ofacceptable structures exists for this portion of the subject biosensor(the chemical shifts or relaxation times for the active-nucleus need tomaintained as detectable).

[0052] Further, it is stressed that the second binding region (20, 21,22, and 23 in FIGS. 1-6) in the targeting carrier comprises that portionof the subject biosensor that interacts with the targetspecies/substrate/molecule/analyte. The first and second binding regionsmay be essentially identical, overlapping, or coextensive or separatedby a plurality of atoms.

[0053] Clearly, the embodiments structures depicted in FIGS. 1-8 for thebasic subject biosensor may contain additional usefulcomponents/structures. As seen in FIGS. 9A and 9B, one, or more.“tether” or “linker” or “spacer” regions 50 may be included in thebiosensor 6. Specifically, FIG. 9A shows a biosensor comprising a firstbinding region 15 for the active-nucleus, a bound active-nucleus 10, asecond binding region 20 for the target, and a tether 50. The tether 50serves to separate the first 15 and second 20 binding regions and mayserve as a site where chemical modification can occur. FIG. 9Billustrates the binding of the second binding group 20 with a target 25.The chemical nature of the tether may be varied and includespolymethylenes, homo and heteropolymers, polyethers, amides, variousfunctional group combinations, amino acids, carbohydrates, and the like.If desired, a plurality of tethered second binding groups may be boundto a first binding region, with each tether and/or second binding groupthe same or different.

[0054] The tether may be derivatized to include a solubilizing region orother desired chemical feature such as additional binding sites and thelike. The solubilizing region aids in solubilizing the biosensor ineither a hydrophilic or hydrophobic environment. It is noted that asolubilizing region may also be included, either in addition to orseparately, in the first and/or second binding regions. A watersolubilizing region may include generally hydrophilic groups such aspeptides, carbohydrates, alcohols, amines, and the like (for a specificexample see FIGS. 11 and 12).

[0055]FIG. 10 illustrates a subject biosensor in which the signal isenhanced by a rapid chemical exchange of the active-nuclei. Freeactive-nuclei 11 rapidly exchange with the active-nucleus 10 bound inthe first binding region 15 to produce an overall increase insensitivity by enhancing the signal.

[0056] More specifically, a functionalized active-nucleus biosensor isdisclosed that capitalizes on the enhanced signal to noise, spectralsimplicity, and chemical shift sensitivity of a hyperpolarized xenon todetect specific biomolecules at the level of tens of nanomoles. Opticalpumping (6) has enhanced the use of xenon as a sensitive probe of itsmolecular environment (7,8). Laser-polarized xenon has been utilized asa diagnostic agent for medical magnetic resonance imaging (MRI) (9) andspectroscopy (10), and as a probe for the investigation of surfaces andcavities in porous materials and biological systems. As indicated for anactive-nucleus, xenon provides information both through directobservation of its NMR spectrum (11-17) and by the transfer of itsenhanced polarization to surrounding spins (18,19). In a proteinsolution, weak xenon-protein interactions render the chemical shift ofxenon dependent on the accessible protein surface, and even allow themonitoring of the protein conformation (20). In order to utilize xenonas a specific sensor of target molecules the xenon was functionalizedfor the purpose of reporting specific interactions with the moleculartarget.

[0057] Specifically, a laser polarized xenon was “functionalized” by abiotin-modified supramolecular cage to detect biotin-avidin binding,thus, the specific target is avidin. Although, as previously indicated,the first binding region that holds the active-nucleus may be one ofmany possible structures, one suitable first binding region or cage is amember of the cryptophane family. Cryptophane has the followingstructure:

[0058] Wherein n=2 for cryptophane-A or n=3 for cryptophane-E.

[0059]FIG. 11 (showing Formula II) depicts a specific targeting carrierin which the first binding region cryptophane-A 15 is covalentlyattached to a tether 50, having a solubilizing region 55, and biotin asthe second binding region 20 (see the Example #1 below for synthesisdetails). The solubilizing region comprises a short peptide chain(Cys-Arg-Lys-Arg) having positively charged groups at physiological pHvalues.

[0060]FIG. 12 (showing Formula III) shows the functionalizedactive-nucleus biosensor when the xenon 10 is bound within the firstbinding region cryptophane-A 15 cage.

EXPERIMENTAL EXAMPLE #1 Functionalized Xenon as a Biosensor

[0061] By way of example and not by way of limitation, one embodiment ofthe subject invention comprises a functionalized system that exhibitsmolecular target recognition. FIGS. 11 (without xenon) and 12 (withxenon) show a biosensor molecule designed to bind both xenon andprotein. Analogous to the general schematic diagrams seen in FIGS. 9Aand 9B, the specifically synthesized subject biosensor molecule consistsof three parts: the cage 15, which contains the xenon 10; the ligand 20,which directs the functionalized xenon 10 to a specific protein; and thetether 50, which links the ligand 20 and the cage 15. In this molecule,it is expected that the binding of the ligand 20 to the target protein(as in analogous FIG. 9B) will be reflected in a change of the xenon NMRspectrum.

[0062] The biotin (ligand second binding region 20) and avidin (targetspecies) couple was chosen because of its high association constant(˜10¹⁵ M⁻¹) (21) and the extensive literature characterizing bindingproperties of modified avidin or biotin (22). The cage 15 chosen forthis embodiment was a cryptophane-A molecule (23) with a polar peptidechain (solubilizing region 55) attached in order to make thecryptophane-A water-soluble.

[0063] The cryptophane-A-based biosensor molecule was synthesized by amodified template directed procedure (23). Starting from3,4-dihydroxybenxaldehyde and using allyl bromide to reversibly protectthe meta-hydroxyl group (24), one of the 6 methoxyl groups incryptophane-A was regioselectively replaced with a free hydroxyl group.Upon reacting with methyl bromoacetate followed by hydrolysis (25), thehydroxyl group in the modified cryptophane-A was converted to acarboxylic acid, which was subsequently coupled (using HOBt/HBTU/DIEAactivation method) to the amino-terminus of a protected short peptideCysArgLysArg on rink amide resin. The resulting cryptophane-A-peptideconjugate was deprotected and cleaved off the resin using “Reagent K”(26), followed by purification with RP-HPLC (MicrosorbTM 80210C5, RP-C18column, flow 4.5 ml/min, buffer A: 0.1% TFA in H₂O, buffer B: 0.1% TFAin CH₃CN, linear gradient from 40% to 80% buffer B in 30 min). Thepurified conjugate was reacted with EZ-linkTMPEO-Maleimide activatedbiotin (Pierce) to give the desired functionalized water-solublecryptophane-A, which was further purified by RP-HPLC (same conditions).The last two peptide conjugated-products were verified bymatrix-assisted laser desorption/ionization (MALDI)-time of flight-(TOF)mass spectrometry. All other intermediates were confirmed by ¹H NMR andMALDI-Fourier Transform mass spectrometry (FTMS).

[0064] Cryptophane-A has been shown to bind xenon with a bindingconstant K≈10³ M⁻¹ in organic solvents (15) but the affinity is likelyto increase in aqueous solution because of the hydrophobic nature ofxenon. The characteristic chemical shift for xenon inside acryptophane-A molecule is very unusual for xenon dissolved in solution,approximately 130 ppm upfield from that of xenon in water. The onlybackground xenon signal in the sample arises from xenon free in solvent,so the signal from the functionalized xenon is easily distinguishable.In the design of a xenon biosensor, a separate peak corresponding toxenon encapsulated by the cage is necessary, requiring both strongbinding and a large difference between the xenon chemical shifts in thecage and solvent environments. The spin-lattice relaxation time for thefunctionalized xenon described herein was measured to be greater than 40s, sufficient time for the required transfer, mixing, and detection ofthe polarized xenon.

[0065] The biosensor solution was prepared by dissolving ˜0.5 mg of thecryptophane derivative (M.W.=2008 g mol⁻¹) in 700 μL of D₂O, yielding aconcentration of ˜300 μM. This concentration was consistent withabsorbance measurements at 284 nm (ε₂₈₄=36,000 M⁻¹cm⁻¹, determined forunmodified cryptophane-A by successive dilutions of a solution of knownconcentration). Approximately 80 nmol of affinity purified egg whiteavidin (Sigma) was used without further purification. Only half of thesample was located inside the detection region, so spectra actuallyreflect detection of ˜40 nmol avidin monomer. Natural abundance xenon(Isotec) was polarized and introduced to the sample using previouslydescribed methods (16), showing ˜5% polarization for the spectra shownin FIGS. 13 and 14. All NMR spectra displayed were obtained in singleacquisition experiments at a nominal ¹²⁹Xe frequency of 82.981 MHz.

[0066]FIG. 13 shows the full ¹²⁹Xe NMR spectrum of the functionalizedxenon in the absence of protein (the trace running near the bottom axisand having a far left peak and far right peaks). The far left peak at193 ppm corresponds to xenon free in water while the far right peaksaround 70 ppm are associated with xenon-bound cryptophane-A. The farright peaks are shown expanded in the center of FIG. 13, where the moreintense, upfield peak (˜70.7 ppm) corresponds to functionalized xenonand has a linewidth of 0.15 ppm (shown by the generalized schematicmodel, as seen in FIG. 9A). A smaller, middle peak (˜71.5 ppm)approximately 1 ppm downfield of the functionalized xenon peak isattributed to xenon bound to a bare cage, without linker and ligand. Asthe unfunctionalized caged xenon does not interact specifically with theprotein, it serves as a useful reference for the chemical shift andsignal intensity of the functionalized xenon in the binding event.

[0067] Upon addition of ˜80 nmol of avidin monomer, a third peak (˜73ppm) appears approximately 2.3 ppm downfield of the functionalized xenonpeak, attributable to functionalized xenon bound to the protein.Correspondingly, the peak assigned to free functionalized xenondecreases in intensity relative to the reference peak while its positionremains unchanged. The peak (˜73 ppm) observed upon the addition ofavidin is an unambiguous identifier of biotin-avidin binding, and hencethe presence of avidin in solution.

[0068] The mechanism of the chemical shift change upon binding mayresult from actual contact between the cryptophane cage and the protein,leading to cage deformation and distortion of the xenon electron cloud.Changes in the rotational and vibrational motions of the cryptophanecage caused by binding to the protein could also affect the xenonchemical shift. Indeed, the sensitivity of xenon to perturbations of thefirst binding region cage is so great that deuteration of one methylgroup results in a readily discernible change in the bound xenonchemical shift (17).

[0069] The subject methodology described herein offers the capability ofmultiplexing by attaching different second binding regions ligands todifferent first binding region cages, forming xenon sensors associatedwith distinct, resolved chemical shifts. As an example of this featureof the subject invention, FIG. 14 shows the changes in bound xenonchemical shift caused by using two different first binding region cages.The top spectrum A is that of xenon bound to cryptophane-A (n=2 inFormula 1 above) in a tetrachloroethane solution and the lower spectrumB is that of xenon bound to cryptophane-E (n=3 in Formula 1 above),similar to cryptophane-A, but with an additional methylene group addedto each of the bridges between the caps. The resulting bound xenonchemical shift is ˜30 ppm upfield from that of xenon bound tocryptophane-A. The linewidths for cryptophanes A and E are broadened bythe exchange of xenon between the cage and tetrachloroethane, theorganic solvent used.

[0070] The diagram in FIG. 15 indicates schematically a multiplexingsystem (multiple functionalized xenon biosensors) for protein assay orscreening procedures. The binding event assay/screening procedures wouldbe distributed over a large chemical shift range by attaching eachsecond binding region ligand to a different first binding region cage.In the absence of the targeted proteins, the spectrum, depicted in FIG.15, would consist of three resolved xenon resonances because of theeffect on the xenon chemical shift caused by cage modifications. Uponbinding each of the targeted proteins, the xenon peaks should shift“independently,” signaling each binding event and reporting theexistence of and amount of protein present. As long as the differencesin shift between xenon in the different cages exceed the shift changeupon binding, it should be possible to monitor and assign multiplebinding events. In FIG. 15, the top spectrum shows the three distinctfunctionalized xenon peaks, corresponding to different cages linked tothree ligands. The bottom spectrum shows the effect of adding thefunctionalized xenon to an unknown solution. Upon addition to theunknown solution, the leftmost peak shifts entirely, representing thecase in which all functionalized xenon is bound to its correspondingprotein. The central peak decreases in intensity and a peakcorresponding to the protein-bound functionalized xenon appears. Therightmost peak remains unaffected, indicating the absence of thecorresponding protein target.

[0071] Thus, enabling experimental data for the subject functionalizedactive-nucleus biosensor has been disclosed that exploits the chemicalshift of functionalized xenon upon binding to a targetspecies/substrate/molecule/analyte. The approach has several criticaladvantages over aspects of current biosensors, in that multiplexingassays and both heterogeneous and homogenous assays are possible.Furthermore, this methodology can be performed in biological materialsin vitro or in vivo by combining the spatial encoding capabilities ofMRI with the biosensing NMR capabilities of the functionalized xenonsensor. As indicated above, potential targets include, are not limitedto, metabolites, proteins, toxins, nucleic acids, and protein plaques.It must be stated that, given the basic information presented herein,refinements of the subject functionalized detector molecules/sensors andthe NMR procedures disclosed herein should further enhance the presentedsensitivity by orders of magnitude, relative to the experimental exampledescribed herein and are within the realm of this disclosure.

[0072] The invention has now been explained with reference to specificembodiments. Other embodiments will be suggested to those of ordinaryskill in the appropriate art upon review of the present specification.

[0073] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

REFERENCES

[0074] All of the following references are herein incorporated byreference. In particular, reference 16 (S. M. Rubin, M. M. Spence, B. M.Goodson, D. E. Wemmer, A. Pines, Proceedings of the National Academy ofSciences of the United States of America 97, 9472-9475 (2000)) was inthe original subject Provisional Application and is specificallyincorporated herein by reference, as are all of the ProvisionalApplication file documents.

[0075] 1. M. Malmqvist, Nature 361, 186-187 (1993).

[0076] 2. W. J. Checovich, R. E. Bolger, T. Burke, Nature 375, 254-256(1995).

[0077] 3. A. Miyawaki et al., Nature 388, 882-887 (1997).

[0078] 4. S. B. Shuker, P. J. Hajduk, R. P. Meadows, S. W. Fesik,Science 274, 1531-1534 (1996).

[0079] 5. A. Y. Louie et al., Nature Biotechnology 18, 321-325 (2000).

[0080] 6. T. G. Walker, W. Happer, Reviews of Modem Physics 69, 629-642(1997).

[0081] 7. C. I. Ratciffe, Annual reports on NMR spectroscopy 36, 124-208(1998).

[0082] 8. Y. Q. Song, B. M. Goodson, A. Pines, Spectroscopy 14, 26-33(1999).

[0083] 9. M. S. Albert et al., Nature 370, 199-201 (1994).

[0084] 10. J. Wolber, A. Cherubini, M. O. Leach, A. Bifone, MagneticResonance in Medicine 43, 491-496 (2000).

[0085] 11. C. R. Bowers et al., Journal of the American Chemical Society121, 9370-9377 (1999).

[0086] 12. R. F. Tilton, I. D. Kuntz, Biochemistry 21, 6850-6857 (1982).

[0087] 13. M. A. Springuel-Huet, J. L. Bonardet, A. Gedeon, J.Fraissard, Magnetic Resonance in Chemistry 37, S1-S13 (1999).

[0088] 14. M. Luhmer et al., Journal of the American Chemical Society121, 3502-3512 (1999).

[0089] 15. K. Bartik, M. Luhmer, J. P. Dutasta, A. Collet, J. Reisse,Journal of the American Chemical Society 120, 784-791 (1998).

[0090] 16. S. M. Rubin, M. M. Spence, B. M. Goodson, D. E. Wemmer, A.Pines, Proceedings of the National Academy of Sciences of the UnitedStates of America 97, 9472-9475 (2000).

[0091] 17. T. Brotin, A. Lesage, L. Emsley, A. Collet, Journal of theAmerican Chemical Society 122, 1171-1174 (2000).

[0092] 18. G. Navon et al., Science 271, 1848-1851 (1996).

[0093] 19. C. Landon, P. Berthault, F. Vovelle, H. Desvaux, ProteinScience 10, 762-770 (2001).

[0094] 20. S. M. Rubin et al., submitted to Journal of the AmericanChemical Society, (2001).

[0095] 21. P. C. Weber, D. H. Ohlendorf, J. J. Wendoloski, F. R.Salemme, Science 243, 85-88 (1989).

[0096] 22. M. Wilchek, E. A. Bayer, Methods in Enzymology 184, 14-45(1990).

[0097] 23. A. Collet, Tetrahedron 43, 5725-5759 (1987).

[0098] 24. S. N. Kilenyi, J. M. Mahaux, E. Vandurme, Journal of OrganicChemistry 56, 2591-2594 (1991).

[0099] 25. J. Canceill, A. Collet, G. Gottarelli, P. Palmieri, Journalof the American Chemical Society 109, 6454-6464 (1987).

[0100] 26. D. S. King, C. G. Fields, G. B. Fields, International Journalof Peptide and Protein Research 36, 255-266 (1990).

What is claimed is:
 1. A macromolecule or molecular complex for use inassaying and screening for a biological target species or environmentwhich: a) contains a magnetically active nucleus; b) is capable ofbinding the biological target species; and c) gives rise to a magneticresonance signal with a unique magnetic resonance property that: i)occurs or changes with the occurrence of said binding event between themacromolecule or molecular complex and the biological target speciesand/or ii) occurs or changes with a subsequent change in the environmentof the biological target species after said binding occurs.
 2. Themacromolecule or molecular complex according to claim 1, wherein saidbinding to the biological target species is either in vivo or in vitro.3. The macromolecule or molecular complex according to claim 1, whereinsaid macromolecule or molecular complex includes a structure selectedfrom a group consisting of monoclonal antibodies, dendrimers,self-assembled lipid complexes, liposomes, cyclodextrins, cryptands,cryptophanes, carcerands, microbubbles, micelles, vesicles, fullerenes,and molecular cage structures.
 4. The macromolecule or molecular complexaccording to claim 1, wherein the macromolecule or molecular complexcomprises a magnetically active gas contained within a molecularcarrier.
 5. The macromolecule or molecular complex according to claim 4,wherein said magnetically active gas is selected from a group consistingof hyperpolarized xenon, sulfur hexafluoride, and hyperpolarized helium.6. The macromolecule or molecular complex according to claim 1, whereinthe macromolecule or molecular complex contains a self-assembled lipidcomplex.
 7. The macromolecule or molecular complex according to claim 6,wherein said self-assembled lipid complex is a liposome.
 8. Themacromolecule or molecular complex according to claim 1, wherein themacromolecule or molecular complex is a rapidly exchanging complexbetween a macromolecule and a magnetically active gas.
 9. Themacromolecule or molecular complex according to claim 8, wherein saidmagnetically active gas is selected from a group consisting ofhyperpolarized xenon, sulfur hexafluoride, and hyperpolarized helium.10. The complex according to claim 8, wherein said macromolecule isselected from a group consisting of cyclodextrins, cryptands,cryptophanes, carcerands, fullerenes, and molecular cage structures. 11.The macromolecule or molecular complex according to claim 1, whereinsaid unique magnetic resonance property is selected from a groupconsisting of chemical shifts and relaxation times.
 12. Themacromolecule or molecular complex according to claim 1, wherein saidchange in environment of the target species includes a change in pH, ionconcentration, or concentration of other molecules near the targetspecies.
 13. A functionalized active-nucleus complex that selectivelyassociates with a biological target species, wherein the functionalizedactive-nucleus complex comprises: a) an active-nucleus and b) atargeting carrier comprising: i) a first binding region having at leasta minimal transient binding of said active-nucleus to form thefunctionalized active-nucleus complex that produces a detectable signalwhen the functionalized active-nucleus complex associates with thetarget species and ii) a second binding region that selectivelyassociates with the target species.
 14. A functionalized active-nucleuscomplex according to claim 13, wherein the functionalized active-nucleuscomplex is selected from a group consisting of a nuclear magneticresonance reporter species and a magnetic resonance imaging contrastagent.
 15. A functionalized active-nucleus complex according to claim13, wherein said active-nucleus is selected from a group consisting ofhyperpolarized xenon, sulfur hexafluoride, ¹⁹F derivatives, andhyperpolarized helium.
 16. A functionalized active-nucleus complexaccording to claim 13, wherein said targeting carrier includes astructure selected from a group consisting of monoclonal antibodies,dendrimers, self-assembled lipid complexes, liposomes, cyclodextrins,cryptands, cryptophanes, carcerands, microbubbles, micelles, vesicles,fullerenes, and molecular cage structures.
 17. A functionalizedactive-nucleus complex according to claim 13, wherein said secondbinding region and said first binding region are coextensive oressentially the same structure.
 18. A functionalized active-nucleuscomplex according to claim 13, wherein: a) said active-nucleus compriseshyperpolarized xenon and b) said first binding region comprises acryptophane.
 19. A functionalized active-nucleus complex according toclaim 18, further comprising a solubilizing region associated with saidtargeting carrier.
 20. A functionalized active-nucleus complex accordingto claim 19, wherein said solubilizing region comprises a moiety thatenhances the solubility of the functionalized active-nucleus complex ina desired environment.
 21. A functionalized active-nucleus complexaccording to claim 19, wherein said solubilizing region comprises atleast one amino acid.
 22. A functionalized active-nucleus complexaccording to claim 18, further comprising a tether connecting said firstand second binding regions.
 23. A functionalized active-nucleus complexaccording to claim 19, wherein said solubilizing region comprises amoiety bound to said tether.
 24. A functionalized active-nucleus complexthat selectively associates with a biomolecular target species, whereinthe functionalized active-nucleus complex comprises: a) anactive-nucleus and b) a targeting carrier comprising: i) a first bindingregion having at least a minimal transient binding of saidactive-nucleus to form the functionalized active-nucleus complex thatproduces a detectable signal when the functionalized active-nucleuscomplex associates with the target species; ii) a second binding regionthat selectively associates with the target species; and iii) a tetherregion connecting said first and said second binding regions.
 25. Afunctionalized active-nucleus complex according to claim 24, wherein thefunctionalized active-nucleus complex is selected from a groupconsisting of a nuclear magnetic resonance reporter species and amagnetic resonance imaging contrast agent.
 26. A functionalizedactive-nucleus complex according to claim 24, wherein saidactive-nucleus is selected from a group consisting of hyperpolarizedxenon, sulfur hexafluoride, polyfluorinated derivatives, andhyperpolarized helium.
 27. A functionalized active-nucleus complexaccording to claim 24, wherein said targeting carrier includes astructure selected from a group consisting of monoclonal antibodies,dendrimers, self-assembled lipid complexes, liposomes, cyclodextrins,cryptands, cryptophanes, carcerands, microbubbles, micelles, vesicles,fullerenes, and molecular cage structures.
 28. A functionalizedactive-nucleus complex according to claim 24, wherein: a) saidactive-nucleus comprises hyperpolarized xenon; b) said first bindingregion comprises a cryptophane; and c) said second binding regioncomprises biotin.
 29. A functionalized active-nucleus complex accordingto claim 24, further comprising a solubilizing region associated withsaid tether region.
 30. A functionalized active-nucleus complexaccording to claim 29, wherein said solubilizing region comprises amoiety that enhances the solubility of the functionalized active-nucleuscomplex in a desired environment.
 31. A functionalized active-nucleuscomplex according to claim 29, wherein said solubilizing regioncomprises at least one polar group.
 32. A functionalized active-nucleuscomplex that selectively associates with at least one biological targetspecies, wherein the functionalized active-nucleus complex comprises: a)an active-nucleus and b) a targeting carrier comprising: i) a firstbinding region having at least a minimal transient binding of saidactive-nucleus to form the functionalized active-nucleus complex thatproduces a detectable signal when the functionalized active-nucleuscomplex associates with the target species; ii) a plurality of secondbinding regions, wherein each of said second binding regions selectivelyassociates with a target species; and iii) a plurality of tether regionswherein said first binding region is connected to each of said secondbinding regions by one of said plurality of said tether regions.
 33. Amethod for assaying and screening for a biological target species whichcomprises: a) functionalizing a magnetically active nucleus byincorporating said nucleus into a macromolucular or molecular complexthat is capable of binding the target species; b) bringing saidmacromolecular or molecular complex into contact with the targetspecies; and c) detecting the occurrence of or change in the nuclearmagnetic resonance signal from said functionalized nucleus in order to:i) monitor the occurrence of binding between said macromolecular ormolecular complex and said target species and/or ii) monitor asubsequent change in the environment of the target species after saidbinding occurs.
 34. The method according to claim 33, wherein saidbinding to said target species is either in vivo or in vitro.
 35. Themethod according to claim 33, wherein said macromolecule or molecularcomplex includes a structure selected from a group consisting ofmonoclonal antibodies, dendrimers, self-assembled lipid complexes,liposomes, cyclodextrins, cryptands, cryptophanes, carcerands,microbubbles, micelles, vesicles, fullerenes, and molecular cagestructures.
 36. The method according to claim 33, wherein saidmacromolecular molecular complex includes a magnetically active gascontained within a molecular carrier.
 37. The method according to claim36, wherein said magnetically active gas is selected from a groupconsisting of hyperpolarized xenon, sulfur hexafluoride, andhyperpolarized helium.
 38. The method according to claim 33, whereinsaid magnetically active gas is selected from a group consisting ofhyperpolarized xenon, sulfur hexafluoride, hyperpolarized helium. 39.The method according to claim 33, wherein said monitoring comprisesdetecting the occurrence of or change in a magnetic resonance signalwith a unique magnetic resonance property.
 40. The method according toclaim 39, wherein said magnetic resonance property is selected from agroup consisting of chemical shifts and relaxation times.
 41. The methodaccording to claim 33, wherein said change in environment of thebiomolecular target comprises a change in pH, ion concentration, orconcentration of other molecules near said target species.
 42. A methodfor assaying and screening for a plurality of biological target speciesutilizing a plurality of functionalized active-nucleus complexes with atleast two of the functionalized active-nucleus complexes having anattraction affinity to different corresponding biological targetspecies, comprising the steps: a) for each functionalized active-nucleuscomplex, functionalizing an active-nucleus by incorporating saidactive-nucleus into a macromolucular or molecular complex that iscapable of binding one of said target species; b) bringing saidmacromolecular or molecular complexes into contact with the targetspecies; and c) detecting the occurrence of or change in a nuclearmagnetic resonance signal from each of said active-nuclei in each ofsaid functionalized active-nucleus complexes in order to: i) monitor theoccurrence of binding between each of said functionalized active-nucleuscomplexes and said target species and/or ii) monitor a subsequent changein the environment of the target species after said binding occurs. 43.The method according to claim 42, wherein said binding to said targetspecies is either in vivo or in vitro.
 44. The method according to claim42, wherein said functionalized active-nucleus complexes includestructures selected from a group consisting of monoclonal antibodies,dendrimers, self-assembled lipid complexes, liposomes, cyclodextrins,cryptands, cryptophanes, carcerands, microbubbles, micelles, vesicles,fullerenes, and molecular cage structures.
 45. The method according toclaim 42, wherein each said functionalized active-nucleus complexincludes a magnetically active gas contained within a molecular carrier.46. The method according to claim 45, wherein said magnetically activegas is selected from a group consisting of hyperpolarized xenon, sulfurhexafluoride, and hyperpolarized helium.
 47. The method according toclaim 42, wherein said monitoring comprises detecting the occurrence ofor change in a magnetic resonance signal with a unique magneticresonance property from each said functionalized active-nucleus complex.48. The method according to claim 47, wherein said magnetic resonanceproperty is selected from a group consisting of chemical shifts andrelaxation times.
 49. The method according to claim 42, wherein saidchange in environment of the biomolecular target comprises a change inpH, ion concentration, or concentration of other molecules near saidtarget species.
 50. A method for assaying and screening for one or morebiological target species which comprises: a) functionalizing amagnetically active nucleus by incorporating said nucleus into amacromolucular or molecular complex that is capable of binding thetarget species; b) bringing said macromolecular or molecular complexinto contact with the target species; and c) detecting the occurrence ofor change in the nuclear magnetic resonance signal from saidfunctionalized nucleus in order to: i) monitor the occurrence of bindingbetween said macromolecular or molecular complex and said target speciesand/or ii) monitor a subsequent change in the environment of the targetspecies after said binding occurs.
 51. A biosensor, comprising: a) anenvironment targeting agent having an attraction affinity to a chemicalenvironment; and b) an active-nucleus carried by said environmenttargeting agent, wherein said environment targeting agent is capable ofrecognizing a change in said chemical environment and a detectablesignal from said active-nucleus indicates said change in said chemicalenvironment.
 52. A biosensor according to claim 51, wherein saidenvironment targeting agent comprises an active-nucleus binding regionfor carrying said active-nucleus and an environment recognition region,wherein said active-nucleus binding region is selected from a groupconsisting essentially of monoclonal antibodies, dendrimers,self-assembled lipid complexes, liposomes, cyclodextrins, cryptands,carcerands, microbubbles, micelles, vesicles, fullerenes, and generalmolecular cage structures.
 53. A biosensor according to claim 51,wherein said active-nucleus is selected from a group consistingessentially of hyperpolarized xenon, sulfur hexafluoride, andhyperpolarized helium.
 54. A biosensor according to claim 51, whereinrecognition of said chemical environment by said environment targetingagent produces a detectable chemical shift from said active-nucleus. 55.A biosensor according to claim 51, wherein recognition of said chemicalenvironment by said environment targeting agent produces a magneticresonance signal.
 56. A biosensor according to claim 51, wherein saidchange in said chemical environment is selected from a group consistingof ion channel functioning, neuron functioning, ion binding andtransport, and oxygen distribution.
 57. A biosensor mixture, comprisinga plurality of functionalized active-nucleus complexes, at least two ofthe functionalized active-nucleus complexes having an attractionaffinity to different corresponding target species, wherein each of saidfunctionalized active-nucleus complexes comprises: a) an active-nucleusand b) a targeting carrier comprising: i) a first binding region havingat least a minimal transient binding of said active-nucleus to form thefunctionalized active-nucleus complex that produces a detectable signalwhen the functionalized active-nucleus complex associates with thetarget species and ii) a second binding region that selectivelyassociates with the target species.
 58. A biosensor mixture according toclaim 57, wherein each of the functionalized active-nucleus complexes isselected from a group consisting of a nuclear magnetic resonancereporter species and a magnetic resonance imaging contrast agent.
 59. Abiosensor mixture according to claim 57, wherein each of saidactive-nuclei is selected from a group consisting of hyperpolarizedxenon, ¹⁹F derivatives, sulfur hexafluoride, and hyperpolarized helium.60. A biosensor mixture according to claim 57, wherein each of saidtargeting carriers includes a structure selected from a group consistingof monoclonal antibodies, dendrimers, self-assembled lipid complexes,liposomes, cyclodextrins, cryptands, cryptophanes, carcerands,microbubbles, micelles, vesicles, fullerenes, and molecular cagestructures.
 61. A biosensor mixture according to claim 57, wherein eachof said second binding regions and said first binding regions arecoextensive or essentially the same structure.
 62. A biosensor mixtureaccording to claim 57, wherein: a) said active-nucleus compriseshyperpolarized xenon and b) said first binding region comprises acryptophane.
 63. A biosensor mixture according to claim 57, wherein eachsaid targeting carrier further comprises a solubilizing regionassociated with each said targeting carrier.
 64. A biosensor mixtureaccording to claim 63, wherein each said solubilizing region comprises amoiety that enhances the solubility of the functionalized active-nucleuscomplex in a desired environment.
 65. A biosensor mixture to claim 64,wherein each said solubilizing region comprises at least one amino acid.66. A biosensor mixture according to claim 57, wherein each saidfunctionalized active-nucleus complex further comprises a tetherconnecting said first and second binding regions.
 67. A biosensormixture according to claim 66, wherein each said functionalizedactive-nucleus complex includes a solubilizing region bound to saidtether.
 68. A biosensor mixture, comprising: a) a plurality offunctionalized active-nucleus complexes, at least two of saidfunctionalized active-nucleus complexes having an attraction affinity todifferent corresponding chemical environments and b) an active-nucleuscarried by each of said functionalized active-nucleus complexes, whereineach said active-nucleus produces a detectable signal in said chemicalenvironment.
 69. A biosensor mixture according to claim 68, wherein eachsaid functionalized active-nucleus complexes includes a targetingcarrier that is selected from a group consisting of monoclonalantibodies, dendrimers, self-assembled lipid complexes, liposomes,cyclodextrins, cryptands, cryptophanes, carcerands, microbubbles,micelles, vesicles, fullerenes, and general molecular cage structures.70. A biosensor mixture according to claim 68, wherein each saidactive-nucleus is selected from a group consisting of hyperpolarizedxenon, sulfur hexafluoride, and hyperpolarized helium.
 71. A biosensormixture according to claim 68, wherein said detectable signal is an NMRchemical shift.
 72. A biosensor mixture according to claim 68, whereinsaid detectable signal is a magnetic resonance signal.